Detection of apolipoprotein B100 early conformational changes during oxidation

Conformational changes of human plasma apolipoprotein B100 (apoB) during oxidative modification of low-density lipoproteins (LDL) have been investigated. Emphasis has been put on the early stages of LDL oxidation and the modification of apoB. We have applied two different modes of LDL oxidation initiation in order to approach the problem from different perspectives. To study conformational changes of the protein and the phospholipids surface monolayer, we have applied attenuated total reflection infrared as well as fluorescence spectroscopy. We have found for the first time that conformational changes of apoB occur even in the earliest stages of oxidation process and that those are located predominantly in the β-sheet regions. The dynamics of changes has also been described and related to different stages of oxidation. After initial increase in particle surface accessibility and mobility, by entering into the propagation phase of oxidation process, LDL surface accessibility and mobility are decreased. Finally, in the decomposition phase of LDL oxidation, as the particle faces large chemical and physical changes, surface mobility and accessibility is increased again. These observations provide new insights into the modifications of LDL particles upon oxidation.     

Oxidation of low density lipoprotein leads to particle aggregation and altered macrophage recognition.

Oxidized (ox-) low density lipoproteins (LDL) is characterized by the formation of lipid peroxides and their decomposition to reactive aldehydes which covalently link to apoB in LDL. These chemical changes are believed to be responsible for the enhanced recognition of ox-LDL by receptors on macrophages in culture. When oxidation is extensive, particle aggregation also occurs. The aim of this study was to characterize aggregation formation and how this influences the interaction of ox-LDL with macrophages in culture. When LDL was oxidized by incubating at 500 micrograms of protein/ml with 10 microM Cu2+ at 20 degrees C for up to 25 h, time-dependent increases in thiobarbituric acid reactive substances, conjugated diene content, electrophoretic mobility, and fluorescence at 360 excitation/430 emission were found. Particle aggregation increased in parallel with several parameters of oxidation and increased with increasing incubation temperatures and LDL concentrations used. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, apoB fragments of reproducible sizes and higher molecular weight species appeared after mild oxidation of LDL. The percent of total apoB remaining aggregated in sodium dodecyl sulfate was 50-80% at high degrees of oxidation, whereas it was far less in LDL that had been aggregated without chemical modification. This suggested that intermolecular cross-linking of apoB had occurred during oxidation of LDL at high concentrations. Degradation of ox-LDL in mouse peritoneal macrophages (MPM) increased in parallel with the degree of oxidation and with particle aggregation but reached a plateau after 12 h. Results from cross-competition studies in MPM with soluble and insoluble portions of extensively ox-LDL and with acetyl-LDL were consistent with uptake of soluble ox-LDL via both the scavenger receptor and another receptor on MPM, and uptake of the insoluble ox-LDL by an alternative mechanism.     

Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous and hypobromous acids.

Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, catalyzes the oxidation of halides to hypohalous acids. At plasma concentrations of halides, hypochlorous acid (HOCl) is the major strong oxidant produced. In contrast, the related enzyme eosinophil peroxidase preferentially generates hypobromous acid (HOBr). Since reagent and MPO-derived HOCl converts low-density lipoprotein (LDL) to a potentially atherogenic form, we investigated the effects of HOBr on LDL modification. Compared to HOCl, HOBr caused 2-3-fold greater oxidation of tryptophan and cysteine residues of the protein moiety (apoB) of LDL and 4-fold greater formation of fatty acid halohydrins from the lipids in LDL. In contrast, HOBr was 2-fold less reactive than HOCl with lysine residues and caused little formation of N-bromamines. Nevertheless, HOBr caused an equivalent increase in the relative electrophoretic mobility of LDL as HOCl, which was not reversed upon subsequent incubation with ascorbate, in contrast to the shift in mobility caused by HOCl. Similar apoB modifications were observed with HOBr generated by MPO/H(2)O(2)/Br(-). In the presence of equivalent concentrations of Cl(-) and Br(-), modifications of LDL by MPO resembled those seen in the presence of Br(-) alone. Interestingly, even at physiological concentrations of the two halides (100 mM Cl(-), 100 microM Br(-)), MPO utilized a portion of the Br(-) to oxidize apoB cysteine residues. MPO also utilized the pseudohalide thiocyanate to oxidize apoB cysteine residues. Our data show that even though HOBr has different reactivities than HOCl with apoB, it is able to alter the charge of LDL, converting it into a potentially atherogenic particle.     

Different apolipoprotein B breakdown patterns in models of oxidized low density lipoprotein.

Low density lipoprotein (LDL) oxidation is characterized by alterations in biological properties and structure of the lipoprotein particles, including breakdown and modification of apolipoprotein B (apoB). We compared apoB breakdown patterns in different models of minimally and extensively oxidized LDL using Western blotting techniques and several monoclonal and polyclonal antibodies. It was found that copper and endothelial cell-mediated oxidation produced a relatively similar apoB banding pattern with progressive fragmentation of apoB during LDL oxidation, whereas malondialdehyde (MDA)- and hydroxynonenal (HNE) -modified LDL produced an aggregated apoB. It is conceivable that apoB fragments present in copper and endothelial cell oxidized LDL lead to the exposure on the lipoprotein surface of different protein epitopes than in aggregated MDA-LDL and HNE-LDL. Although all models of extensively oxidized LDL led to increased lipid uptake in macrophages, mild degrees of oxidation interfered with LDL uptake in fibroblasts and extensively oxidized LDL impaired degradation of native LDL in fibroblasts. We suggest that in order to improve interpretation and comparison of results, data obtained with various models of oxidized LDL should be compared to the simpliest and most reproducible models of 3 h and 18 h copper-oxidized LDL (apoB breakdown) and MDA-LDL (apoB aggregation) since different models of oxidized LDL have significant differences in apoB breakdown and aggregation patterns which may affect immunological and biological properties of oxidized LDL.     

Comparison of two methods for radioiodination on the oxidizability properties of low density lipoprotein

Radiolabeling of low density lipoprotein (LDL) apoB100 with 125I, an oxidative process, is commonly used in lipoprotein investigation. Since 1) LDL is unstable and oxidation-prone, 2) the modification of apoB100 by oxidation increases the negative charge of particles and leads to the uptake of modified LDL through the scavenger receptor pathway, and 3) oxidized LDL is cytotoxic, it is relevant to investigate whether the oxidative stability of LDL is influenced by its labeling with 125I. The aim of this study was to investigate and compare lipid and protein oxidation markers in human LDL after labeling with 125I by two widely adopted methods that use ICl or the chloramide 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril as the oxidizing agent. Native LDL served as a common control and sham-iodinated LDL as a handling control for each procedure. The resistance against copper-induced oxidation of 125I-LDL labeled with ICl was similar to that of controls with regard to the lag time and maximal amount of conjugated diene formed, as there were levels of initial conjugated diene, alpha-tocopherol, and tryptophan. However, radioiodination with the chloramide accelerated the onset of the rapid phase of LDL oxidation due to a drastic depletion of alpha-tocopherol and increased conjugated diene content. Measurements of copper-induced LDL oxidizability showed enhanced indices of lipid oxidation. The lag time and the time to maximal diene production were 65% and 30% shorter than controls. This was accompanied by a 50% reduced tryptophan fluorescence. The anionic surface charge of the LDL particle increased moderately with both labeling procedures. The results indicate that labeling of LDL with 125I may oxidize lipids and apoB100 to a variable extent, depending on the nature of the iodinating agent. This is why assessment of the oxidizability properties of 125I-labeled LDL is recommended for reliable biological studies.     

Composition and structure of lipopolysaccharide-human plasma low density lipoprotein complex. Analytical ultracentrifugation, 31P-NMR, ESR and fluorescence spectroscopy studies

Complexes of Salmonella typhimurium lipopolysaccharide toxin (LPS) with low density lipoproteins (LDL) prepared in vitro have been analyzed. LPS-LDL complexes were found to comprise approx. 0.24 mg LPS/mg LDL protein. The major protein of complexes was apolipoprotein apoB-100 (greater than or equal to 90-95%). Incorporation of LPS molecules into LDL was accompanied by small changes in lipid composition, i.e. the phosphatidylcholine content was diminished by approx. 11% and the free fatty acid concentration was raised 2-fold. Analytical ultracentrifugation showed that insertion of LPS into LDL results in the increase of a portion of particles with higher density (lower flotation coefficient) compared to initial LDL. As was evidenced by ESR, in LPS-LDL complexes, the phospholipid hydrocarbon chains are more ordered than in LDL. 31P-NMR spectra indicated that in LPS-LDL complexes the mobility of phospholipid polar headgroups is restricted in comparison with LDL. Application of the shift reagent (Pr3+) revealed that phospholipid molecules form a monolayer structure on the surface of complexes. Upon binding of LPS to LDL, a maximum of the apoB intrinsic fluorescence was slightly red-shifted (1-2 nm) which may testify that the localization of apoB remains nearly unchanged. For LPS-LDL complexes, the accessibility of apoB fluorophores to quenchers (I-, Cs+, acrylamide) did not dramatically differ from that of LDL. It is concluded that rather large amounts of LPS (about 9-10 molecules) can accommodate in one LDL particle without severely perturbing its original composition and structure. Moreover, in the LPS-LDL complexes, oligosaccharide chains of LPS screen notably neither phospholipid polar headgroups nor, what is very important, apoB. LPS-LDL complexes are suggested to be able in vivo to bind to cellular apoB/E receptors, possible LPS receptors and scavenger-receptors of macrophages (monocytes).     

An immunochemical marker of low density lipoprotein oxidation

Using monoclonal antibodies against apolipoprotein B (apoB) we studied changes in apoB immunoreactivity during copper ion-mediated oxidation of human low density lipoprotein (LDL). The radioimmunoassay experiments demonstrated the decrease of immunoreactivity of three different epitopes of apoB located in different parts of the protein; at the same time the immunoreactivity of another epitope, previously mapped to the C-terminal 20 amino acids of apoB increased markedly during the first 6 h of LDL oxidation and diminished gradually upon prolonged incubation with copper ions. The fate of LDL during oxidation was also monitored using electrophoretic techniques combined with immunodetection. These experiments showed a rapid fragmentation and disappearance of immunoreactive apoB. They also indicated that the diminishing LDL immunoreactivity detectable during oxidation is associated with apoB fragments still attached to the lipid core. The changes in apoB immunoreactivity during Cu2+ treatment of LDL are similar to those observed upon LDL aging. Therefore, it appears that the enhancement of immunoreactivity of the C-terminus of apoB is a general phenomenon associated with various kinds of oxidative modifications of LDL.     

Paradoxical protective effect of aminoguanidine toward low-density lipoprotein oxidation: inhibition of apolipoprotein B fragmentation without preventing its carbonylation. Mechanism of action of aminoguanidine

Oxidative modification of low-density lipoproteins (LDLs) is an important feature in the initiation and progression of atherosclerosis. Aminoguanidine (AMG), classically described as an inhibitor of advanced glycation end products, turned out to be also efficient in animal models as an antioxidant against lipid peroxidation. The originality of the present study was based on the simultaneous assessment of the oxidation of LDL lipid and protein moieties in order to characterize the molecular sites of AMG protection. Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes (CD) and hydroperoxide molecular species from cholesteryl esters (CEOOH) and phosphatidylcholines (PCOOH). LDL protein oxidative modifications were assessed by evaluating apoB carbonylation and fragmentation. The LDL oxidation was mediated by water gamma radiolysis, which has the advantage of being quantitative and highly selective with regard to the free radicals produced. Here, we reported that AMG resulted in a protection of LDLs against lipid peroxidation (both in the lag phase and in the propagation phase) and against apoB fragmentation in a concentration-dependent manner, due to the scavenging effect of AMG toward lipid peroxyl radicals. Paradoxically, AMG was poorly efficient against apoB carbonylation that began during the lag phase. We hypothesize that, even in the presence of AMG, a nonnegligible proportion of (*)OH radicals remained able to initiate oxidation of the LDL protein moiety, leading to apoB carbonylation.     

Human LDL core cholesterol ester packing: three-dimensional image reconstruction and SAXS simulation studies

Human LDL undergoes a reversible thermal order-disorder phase transition associated with the cholesterol ester packing in the lipid core. Structural changes associated with this phase transition have been shown to affect the resistance of LDL to oxidation in vitro studies. Previous electron cryo-microscopy studies have provided image evidence that the cholesterol ester is packed in three flat layers in the core at temperatures below the phase transition. To study changes in lipid packing, overall structure and particle morphology in three dimensions (3D) subsequent to the phase transition, we cryo-preserved human LDL at a temperature above phase transition (53°C) and examined the sample by electron microscopy and image reconstruction. The LDL frozen from 53°C adopted a different morphology. The central density layer was disrupted and the outer two layers formed a "disrupted shell"-shaped density, located concentrically underneath the surface density of the LDL particle. Simulation of the small angle X-ray scattering curves and comparison with published data suggested that this disrupted shell organization represents an intermediate state in the transition from isotropic to layered packing of the lipid. Thus, the results revealed, with 3D images, the lipid packing in the dynamic process of the LDL lipid-core phase transition.     

Human apo-lipoprotein B from normal plasma contains oxidised peptides

Oxidation of low density lipoprotein (LDL) may be atherogenic, but radical-initiated oxidation of its apoprotein B-100 (apoB) has been little studied. Transition metal ions iron and copper are candidates for mediating radical oxidation of LDL in vivo. Therefore, we studied the copper-ion-induced oxidation of apoB in human LDL. Using HPLC methods developed in our recent work, we studied the destruction of native and the generation of six oxidised amino acids; we also assessed the release of peptides from the LDL particle by FPLC. We observed time-dependent losses of apoB histidine, lysine and glycine. Long-lived reactive species, the reductant DOPA, and the oxidant hydroperoxides of valine and leucine (measured as hydroxides after reduction), were generated. Their relative abundance (mol/mol of parent amino acid) was DOPA>o- and m-tyrosine>dityrosine, valine-hydroxides, leucine hydroxides. Low molecular weight fragments were also released from the LDL in a time-dependent manner, contained hydroperoxides sensitive to GSH peroxidase, and generated radicals on reaction with iron–EDTA. The fragments contained peptides active in the quinone redox cycling procedure, comprising 0.25% of the supplied LDL amino acids. Characteristic peptides were present in each FPLC fraction containing the fragments, as judged by further HPLC fractionation. Some fragments were present in the unoxidised LDL preparations, and when these were largely removed by FPLC, copper oxidation could still generate fragments, suggesting that those present in the starting material might indicate prior oxidation. Concordantly, we found that fresh plasma LDL apoB contained 3% of total plasma protein-bound oxidised amino acids, and with the same relative abundance. We conclude that plasma proteins including apoB are subject to physiological oxidation, similar to that inflicted by copper ions; the latter may contribute to intimal LDL oxidation, which could be the source of oxidised plasma apoB.     

Protein hydroperoxides are a major product of low density lipoprotein oxidation during copper, peroxyl radical and macrophage-mediated oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2'-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble alpha-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

Binding of low density lipoproteins to lipoprotein lipase is dependent on lipids but not on apolipoprotein B

Lipoprotein lipase (LPL) efficiently mediates the binding of lipoprotein particles to lipoprotein receptors and to proteoglycans at cell surfaces and in the extracellular matrix. It has been proposed that LPL increases the retention of atherogenic lipoproteins in the vessel wall and mediates the uptake of lipoproteins in cells, thereby promoting lipid accumulation and plaque formation. We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B (apoB). Chemical modification of lysines and arginines in apoB or mutation of its main proteoglycan binding site did not abolish the interaction of LDL with LPL as shown by surface plasmon resonance (SPR) and by experiments with THP-I macrophages. Recombinant LDL with either apoB100 or apoB48 bound with similar affinity. In contrast, partial delipidation of LDL markedly decreased binding to LPL. In cell culture experiments, phosphatidylcholine-containing liposomes competed efficiently with LDL for binding to LPL. Each LDL particle bound several (up to 15) LPL dimers as determined by SPR and by experiments with THP-I macrophages. A recombinant NH(2)-terminal fragment of apoB (apoB17) bound with low affinity to LPL as shown by SPR, but this interaction was completely abolished by partial delipidation of apoB17. We conclude that the LPL-apoB interaction is not significant in bridging LDL to cell surfaces and matrix components; the main interaction is between LPL and the LDL lipids.     

Protein Hydroperoxides are a Major Product of Low Density Lipoprotein Oxidation During Copper,Peroxyl Radical and Macrophage-mediated Oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble α-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

Decrease in the particle size of low-density lipoprotein (LDL) by oxidation

A radical reaction of low-density lipoprotein (LDL) causes fragmentation and cross-link of apolipoprotein B-100 (apoB). LDL (50 microg/ml) was subjected to the well-studied oxidation with Cu(2+) (1.67 microM). The concentration of alpha-tocopherol decreased to 10% of the initial level during the first 30 min. After this lag time, the conjugated diene content, as measured by absorption at 234 nm, started increasing and the residual apoB at 512 kDa determined by immunoblot after SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) was also decreased. The particle size of LDL determined by nondenaturing gradient gel electrophoresis decreased steadily during the initial 120 min, when residual native apoB was only 30% of the initial level. Plasma was also oxidized with Cu(2+) (400 microM). Under this condition, a clear lag time was not observed and alpha-tocopherol content, apoB, and the LDL particle size were decreased simultaneously. Based on these experiments, we propose that an oxidation reaction is involved in the formation of small dense LDL.     

A lipoprotein-containing particle is transferred from the serum across the mammary epithelium into the milk of lactating mice

To investigate the role of low-density lipoprotein (LDL) in the delivery of cholesterol to the mammary gland during pregnancy and lactation, we examined the distribution of radioactivity from (125)I-tyramine cellobiose-LDL injected into the tail vein of female mice at various stages of the reproductive cycle. Changes in the proportion of isotope taken up by the mammary gland largely reflected the increased weight of the gland in pregnancy and lactation. In addition, during lactation, radioactivity was found in the milk and was associated with a protein of the molecular weight of apoB-100. Quantitatively similar results were obtained with mice homozygous for disruption of the LDL receptor gene (LDLR null). Analysis of endogenous lipoproteins showed that the milk lipoprotein particles were denser than the corresponding serum lipoproteins and largely depleted of triglyceride and cholesterol. Using fluorescence microscopy we visualize the sorting of apoB protein from the LDL lipid phase at the basal surface of the mammary epithelial cell of both wild-type and LDLR-null mice. Our findings provide evidence that the mammary epithelium of the lactating mouse is able to take up LDL from the plasma by a non-LDLR-mediated process. An apoB-containing particle from which the cholesterol has been removed is transferred into milk.     

The influence of the triglyceride content of low density lipoprotein on the interaction of apolipoprotein B-100 with cells

To study the effect of triglyceride content of low density lipoprotein (LDL) on its physicochemical and biological properties, we have depleted the triglyceride by incubation with hepatic lipase (HL-LDL) and raised the triglyceride by incubation of HL-LDL with very low density lipoprotein and lipoprotein-deficient serum. HL-LDL was taken up by human monocyte-derived macrophages and by human skin fibroblasts at an increased rate compared to untreated LDL. Incubation of the various LDL preparations revealed that cellular LDL degradation as well as LDL-mediated cholesterol esterification were inversely related to the triglyceride content of the LDL preparation. Modification of the triglyceride content of LDL also was associated with changes in the free fatty acid content, but the interaction of the LDL with cells was unaffected by the level of this component. The triglyceride content of LDL was found to be reciprocally related to the number of free lysine amino groups of LDL apolipoprotein B (apoB) which could be labeled with trinitrobenzenesulfonic acid. 13C-Nuclear magnetic resonance (NMR) spectra of native LDL and HL-LDL samples containing [13CH3]2 lysine residues formed by reductive methylation (11-13% modification) showed that the arrangement of apoB lysines is perturbed by the exposure to hepatic lipase. The ratio of labeled lysines with pK 8.9 to those with pK 10.5 exposed on the surface of LDL particles was decreased by about 40% by lipase treatment. These effects are apparently due to changes in local apoB conformation because circular dichroism spectra revealed that the average secondary structure of the entire apoB molecule is the same in native LDL and HL-LDL. The triglyceride content of LDL reciprocally affected its binding to a monoclonal antibody which recognizes epitopes around the LDL receptor binding domain of apoB. The above evidence indicates that modulation of the core triglyceride and possibly also surface phospholipid content of LDL can alter the conformation of apoB on the surface of the particle, thereby influencing the interaction with cell surface LDL receptors.     

Protective effects of endomorphins, endogenous opioid peptides in the brain, on human low density lipoprotein oxidation

Neurodegenerative disorders are associated with oxidative stress. Low density lipoprotein (LDL) exists in the brain and is especially sensitive to oxidative damage. Oxidative modification of LDL has been implicated in the pathogenesis of neurodegenerative diseases. Therefore, protecting LDL from oxidation may be essential in the brain. The antioxidative effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain, on LDL oxidation has been investigated in vitro. The peroxidation was initiated by either copper ions or a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes, thiobarbituric acid reactive substances, and the relative electrophoretic mobility. Low density lipoprotein oxidative modifications were assessed by evaluating apoB carbonylation and fragmentation. Endomorphins markedly and in a concentration-dependent manner inhibited Cu2+ and AAPH induced the oxidation of LDL, due to the free radical scavenging effects of endomorphins. In all assay systems, EM1 was more potent than EM2 and l-glutathione, a major intracellular water-soluble antioxidant. We propose that endomorphins provide protection against free radical-induced neurodegenerative disorders.     

Immuno-electron cryo-microscopy imaging reveals a looped topology of apoB at the surface of human LDL

A single copy of apoB is the sole protein component of human LDL. ApoB is crucial for LDL particle stabilization and is the ligand for LDL receptor, through which cholesterol is delivered to cells. Dysregulation of the pathways of LDL metabolism is well documented in the pathophysiology of atherosclerosis. However, an understanding of the structure of LDL and apoB underlying these biological processes remains limited. In this study, we derived a 22 Å-resolution three-dimensional (3D) density map of LDL using cryo-electron microscopy and image reconstruction, which showed a backbone of high-density regions that encircle the LDL particle. Additional high-density belts complemented this backbone high density to enclose the edge of the LDL particle. Image reconstructions of monoclonal antibody-labeled LDL located six epitopes in five putative domains of apoB in 3D. Epitopes in the LDL receptor binding domain were located on one side of the LDL particle, and epitopes in the N-terminal and C-terminal domains of apoB were in close proximity at the front side of the particle. Such image information revealed a looped topology of apoB on the LDL surface and demonstrated the active role of apoB in maintaining the shape of the LDL particle.     

Free radical modification of lipoproteins and cholesterol accumulation in cells upon atherosclerosis.

An electron spin probe study was made of the effect of lipid peroxidation (LPO) on the structure of surface proteolipid layer of human serum low-density lipoproteins (LDL). The results obtained with a positively charged spin label and stearic acid spin probes with doxyl labels at positions 5, 12, and 16 revealed that LPO caused a decrease in phospholipid molecule mobility both in the region of polar heads and in the region of acyl chains till the depth of at least 1.7 mm from water-lipid interface. Under relatively high levels of oxidation (more than 6 mumol MDA/g LDL phospholipid) the polarity of lipid phase increased. The decrease in efficiency of tryptophan fluorescence quenching by nitroxide fragments incorporated in hydrophobic regions at the depth of approximately 2 nm from water-lipid interface indicated that lipid-protein interaction was disturbed as a result of oxidation of LDL lipids. In addition, the LPO-induced modification of apo-B, the main protein of LDL, was examined with maleimide spin label. LPO led to increase in mobility of strongly immobilized maleimide labels and in the number of weakly immobilized ones. Oxidized LDL revealed decreased ability to incorporate spin-labeled steroid (androstane) as compared to native ones. LPO-induced structural changes of LDL surface are supposed to be a reason of enhanced accumulation of cholesterol in human monocytes during their incubation with oxidized LDL. The cholesterol content in red cells was shown to be directly correlated to MDA content in apo-B containing lipoproteins but not in whole serum. Our findings suggest that free radical modification of serum lipoproteins but not solely an increased level of LPO products in blood is one important cause for cholesterol accumulation in cells and, apparently, for their transformation into foam cells during atherosclerosis.     

Modification of human serum low density lipoprotein by oxidation--characterization and pathophysiological implications

Plasma low density lipoprotein (LDL) can undergo free radical oxidation either catalyzed by divalent cations, such as Cu2+ or Fe2+ or promoted by incubation with cultured cells such as endothelial cells, smooth muscle cells and monocytes. The content of vitamin E, beta-carotene and unsaturated fatty acids is decreased in oxidized LDL. A breakdown of apolipoprotein-B (apoB), hydrolysis of the phospholipids, an increase of thiobarbituric acid reactive substances and the generation of aldehydes also occur. Changes in the ratio of lipid to protein, the electrophoretic mobility and the fluorescent properties have also been reported to accompany oxidation of this lipoprotein. The functional changes of oxidized LDL include its recognition by the scavenger receptor on macrophages, its cytotoxicity especially to proliferating cells, its chemotactic properties with respect to monocyte-macrophages and its regulation of platelet-derived growth factor-like protein (PDGFc) production by endothelial cells. In this article we summarize some of the contributions to this topic and present speculations relating oxidized LDL to pathological conditions such as atherosclerosis.